Thursday, 23 October 2014

PCR for food microbiology

Real-time PCR assays for food microbiology have been developed into commercial products whereby some or all of the steps can be automated to minimise the number of operations involved and reduce the risk of contamination. Automation requires a higher capital investment and so will depend on the sample throughput required. The reaction usually takes place inside a computer-controlled combined thermocycler/fluorescence detection instrument and uses pre-prepared reagents. For foodborne pathogen detection tests, the entire process can be completed within 20-30 hours with sufficient sensitivity to detect a single cell in a 25g sample.

The main advantage for PCR-based methods is in shorter detection times, but the high degree of automation built into PCR systems also allows relatively unskilled staff to run them without extensive training. The high specificity of PCR can also mean fewer repeat tests. The principal disadvantage is currently cost, both in terms of capital outlay and consumables. While larger laboratories can benefit from reduced labour costs, economies of scale and rapid results, the benefits for smaller labs may be less clear.

A number of commercial PCR systems are currently offered for food pathogen detection. One of the first into the marketplace was the Bax® system from Dupont Qualicon and this has been joined by TaqMan® and MicroSEQ® food pathogen detection kits from Life Technologies, iQ Check real-time PCR kits from Bio-Rad, foodproof® real-time PCR detection kits distributed by Merck, and several others.

One of the main reasons for the comparatively high capital cost of PCR-based detection systems is the need for thermocycling during the amplification step. Instruments must be capable of very accurate and precise temperature control throughout the cycle. In addition to the capital investment for the instrumentation, the chemistry is also expensive as it uses fluorescent probes. Novel developments in DNA synthesis have demonstrated alternative solutions for DNA amplification under isothermal conditions without the need for a thermocycler. There are several types of nucleic acid amplification technologies. Amongst the isothermal DNA amplification technologies that have been developed, loop-mediated isothermal amplification (LAMP) has been used widely to detect microorganisms and is a promising and suitable technology for the rapid detection of pathogen in the field. LAMP uses multiple primers and a bacterial polymerase, Bst polymerase, derived from Bacillus stereothermophilus to amplify DNA rapidly at a constant 63oC. This does away with the need for a thermocycler component in the instrument and can reduce the cost by up to two thirds.

A commercial isothermal amplification system has already been developed for food pathogen detection. The 3M™ Molecular Detection System uses a unique bioluminescence method to detect the amplification of DNA sequences and is designed to be simple to use. The amplification and detection processes are completed within 75 minutes with real-time positive results available as early as 15 minutes. An overnight single enrichment step is still required at present. Test kits for E. coli O157 including H7, Salmonella and Listeria spp. detection in food and environmental samples are currently available.

Portable PCR-based instruments have also been developed recently. For example, Idaho Technology markets the ‘Ruggedized Advanced Pathogen Identification Device’ or R.A.P.I.D.® System, which uses an air thermocycling process and a fluorimetric detection system to detect Salmonella, Listeria, E. coli O157 and Campylobacter in food samples.
Posted by Rapid Microbiology